Hepes czb medium

Female mice were induced to undergo superovulation via treatment with 5 IU pregnant mare serum gonadotropin (ASKA Pharmaceutical Co., Ltd., Tokyo, Japan) followed 48 hours later by 5 IU human chorionic gonadotropin (hCG) (ASKA Pharmaceutical Co., Ltd.). Sixteen hours after hCG treatment, cumulus–oocyte complexes (COCs) were collected from the oviducts as quickly as possible and placed into HEPES CZB medium containing 0.1% bovine testicular hyaluronidase (Sigma-Aldrich, St Louis, MO, USA). After several minutes, oocytes were washed and placed into a droplet of CZB medium for culture.

Superovulation was conducted as previously described [29 (link)]. Cumulus oocyte complexes (COCs) at the metaphase-II stage were collected from the oviducts of the mice that were superovulated by an i.p. injection of 5 IU equine chorionic gonadotropin (eCG; Nippon Zenyaku Kogyo, Tokyo, Japan) followed by 5 IU human chorionic gonadotropin (hCG; Asuka Pharmaceutical Co., Tokyo, Japan) 48 h later. Twelve to fourteen hours after hCG injection, the females were sacrificed and their oviductal ampullae were removed. The oviductal ampullae were placed in oil, and COCs were collected from the oviductal ampullae and then transferred to HEPES-buffered CZB (HEPES–CZB) medium [30 (link)]. To disperse the cumulus, the COCs were transferred into a 100-μL droplet of HEPES-CZB medium containing 0.1% hyaluronidase (Sigma, St. Louis, MO, USA) for 3 min. The cumulus-free oocytes were washed twice and then moved to a 20-μL droplet of CZB medium [31 (link)] for further experiments.